| Nucleic acid detection,as an important detection method in In Vitro Diagnosis(IVD),is the gold standard for the detection of many pathogens.However,because traditional nucleic acid detection involves different biochemical reaction processes,professionals are required to use different instruments in a professional PCR laboratory.Completion is often time-consuming and laborious.Therefore,this thesis developed an integrated nucleic acid detection system that integrates cell lysis,nucleic acid purification,amplification and detection based on microfluidic technology,and focuses on the pre-processing technology of nucleic acid detection on microfluidic chips.The main work content and conclusions are as follows:(1)Analyzed and compared a variety of different sample pre-processing methods,designed an integrated nucleic acid detection microfluidic chip based on chemical lysis and magnetic bead method as sample pre-processing,and analyzed the magnetic beads in the channel of the chip,changes in capillary force when passing through the capillary passive valve at each stage of the transfer process.(2)According to the designed microfluidic chip and the biochemical reaction steps to be achieved,the entire detection instrument is designed,and different components are designed,including reagent storage and release components,main drive components,temperature control components,permanent magnet array components,fluorescence detection components have constructed a prototype of an integrated nucleic acid detection instrument.(3)The droplet mechanics model containing magnetic beads is established through theoretical analysis.The magnetic field environment of the magnetic bead clusters was simulated with the aid of the finite element simulation platform,and the magnetic bead clusters of different volumes were subjected to different positions.Analyze the influence of the magnetic bead cluster volume,the droplet volume,and the moving speed of the permanent magnet on the movement state of the droplet,and obtain the matching relationship between the droplet volume,the moving speed of the permanent magnet and the magnetic bead cluster volume.Conclude the droplet manipulation diagrams.(4)Quantitative analysis of the liquid carried during the transfer of magnetic beads and by gray value analysis method,and it was estimated that the amount of liquid carried from the lysis pool to the amplification pool was less than 0.1%,which had no effect on the amplification reaction;The recovery rate of the magnetic beads was analyzed using the weighing method.The results showed that the recovery rate of the magnetic beads on the chip was slightly lower than that of the manual method,but the consistency was better than that of the manual method,and the time-consuming was shorter;finally,the different vibration frequencies were analyzed.On the effect of droplet mixing,the results show that when the amplitude is 20mm,the vibration frequency of 2Hz has the best mixing effect.(5)A series of experiments were carried out to evaluate the developed nucleic acid detection integrated microfluidic system.The nucleic acid purification experiment on the chip proved that the purification rate exceeded 55%,reaching a reasonable level;the four pathogens were tested on the chip.The test results of multi-target nucleic acid detection showed that all four pathogens were amplified and there was no cross-infection,which proved the effectiveness of the system;the positive control in the kit determined the lower limit of detection to be 10~2copies/sample,proved that the microfluidic chip can quantitatively analyze the nucleic acid content of the initial sample. |
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